Method for detecting diseases that are associated with defects of cystic fibrosis transmembrane conductance regulator (cftr)protein

ABSTRACT

The invention concerns a method for the diagnosis of membrane channel defects in cells by comparing defective with non-defective cells with the following steps: influencing the volume regulation of the cells; recording the change of volume regulation resulting therefrom in non-defective cells; determining the existence of defective cells with a volume change deviating therefrom, in order to provide a suitable method which enables the identification of diseases based on CFTR defects, in particular cystic fibrosis, at an early stage.

The invention concerns non-genetic verification procedures and means forillnesses that are based on defects of the CFTR, in particular for theillness mucoviscidosis (cystic fibrosis or CF) and claims priority ofthe German patent applications 102 08 293.6 and 102 16 160.7(incorporated herein by reference).

Different diseases based on defects of the CFTR, for example cysticfibrosis, the congenital absence of the vas deferens, and some forms ofpancreatitis, are well-known. Cystic fibrosis—also known asmucoviscidosis—represents one of the most frequently occurringgenetically caused illnesses. The illness appears in regionally varyingfrequency of approx. 1:2500 newborn children.

Mucoviscidosis is autosomal recessive and arises from a defect at thelong arm of chromosome 7. The gene that encodes the protein CFTR (cysticfibrosis transmembrane conductance regulator), a membrane transportprotein, is affected. The main symptom of this serious illness is ageneral lack of function of the epithelia, all exocrine glands, the lungand the digestive tract. The illness expresses itself by an increasedviscosity of the mucous secretions of the glands in the lung and in thepancreas. In the progressive illness, substantial anatomical changesoccur, with the consequence of severe complications in the region of therespiratory system, as for example chronic infections with emphysema ofthe lung and serious disturbances of digestion (malabsorption) withliquid and electrolyte losses. Treatment of cystic fibrosis takes placevia enzyme substitution, specific antibiotic and physiotherapeutictreatment (percussion massage). The illness is not curable.Mucoviscidosis patients suffer from a substantial restriction of theirquality of life and as a rule, reach an age of only approx. 40 yearsdespite today's good medical support.

The disease emergence is not clearly clarified yet. The affected genecodes for a chloride channel located predominantly in the membrane ofepithelial cells. A multiplicity of different mutations, which lead toincreased mucus viscosity and a changed composition of the secretions,is described (Pschyrembel, Clinical Dictionary, 258th Edition). The mostfrequent mutation is the deletion of phenylalanin at position 508(dF508). Beside the homozygote form of the mucoviscidosis, the illnesscan also develop with heterozygote genetic carriers with varyingseverity level and also can develop at an advanced age. About 4% of thewhite population of Europe and the USA carry a heterozygote CFTRmutation.

From the literature it is well-known that with CF patients with thedF508 homozygote mutation, the CFTR protein in the plasma membrane ofthe cells is either not achieved or is insufficiently anchored in themembrane. Such a CFTR protein is then nonfunctioning.

From the literature it is further well-known that the CFTR has asubstantial influence on the regulation of the cell volume. In cells, inwhich the CFTR function is not disturbed, it encroaches on an autocrinemechanism, which is steered by the release of and the signaltransmission by ATP (adenosine triphosphate) through a separate ionchannel. A hypotonic medium causes a water influx directed inward to thecell. The cell swelling resulting from it activates the CFTR that againreleases an ATP transport from the cell. The extracellular ATP activatespurinergic receptors, which energize the phospolipase C (PLC) in theline for the formation of inositol triphosphate (IP₃). The inositoltriphosphate increases the intracellular concentration of calcium ions(Ca²⁺). A increased intracellular calcium ion concentration activatesthe calcium-dependent potassium and chloride channels as well aschannels for osmolytes, for example Taurin. Thereupon potassium andchloride ions leave the cell. Because of this net salt loss water flowsosmotically backwards. As a consequence the cell shrinks (Braunstein, G.B. et al. The Journal of Biological Chemistry, volume 276, No. 9. Issueof March 2, pp. 6621-6630, 2001). This process is designated regulatoryvolume decrease (RVD). It was shown that the release of ATP from humanred blood cells after mechanical deformation is dependent on CFTR.(Sprague R S, Ellsworth M L, Stephenson A H , Kleinhenz M E, Lonigro AJ, deformation induced ATP release from red blood cells requires cysticfibrosis transmembrane conductance regulator activity. American Journalof Physiology, 275:H172M1732, 1988). It was shown that in hypotonicmedia the red blood cells of some species regulate their volumedepending on ATP; however, not human red blood cells. (Light D S, CapesT L, Gronau R T, Adler M R. Extracellular ATP stimulates volume decreasein Necturus red blood cell. American Journal of Physiology, Sep; 277(3Pt 1):C480-01. 1999).

Methods of CF Diagnosis:

1) Until now the diagnosis of cystic fibrosis is accomplished, amongother methods, by a sweat test in which the CFTR dependent chlorideabsorption of the epithelium cells of the sweat ducts is determined bymeasurement of the chloride concentration in the sweat (Patent-Nr.:WO00/13713 title: Macroscopic sweat test for cystic fibrosis). With CFpatients an increased chloride concentration is found. The sweat test isonly practicable starting from the 4^(th) month of life, and frequentlyprovides unclear results. Besides, it is very time consuming and complexfor personnel and thus expensive.

2) Other methods are the measurement of the immunoreactive trypsinintroduced by the pancreatic insufficiency of CF patients into the blood(Patent No.: AU 6445186. Title: Detection of immunoreactive trypsin andcystic fibrosis using monoclonal antibody) and

3) the verification of an increase of the albumin content in themeconium (infant fecal material) of babies (patent No.: U.S. Pat. No.3,902,847, title; Diagnostic Device and Method for the Diagnosis ofmucoviscidosis (cystic fibrosis)).

4) In addition, CF diagnosis can be made by the investigation ofsalivary gland secretion and by

5) the exact, but complex, and thus so far little applied,transepithelial nasal potential measurement of the nasal mucus membrane.

6) A further possibility for the diagnosis of cystic fibrosis exists inthe recognition of the mutation by direct gene analysis (Patent-No.;W094/15216. title: Detection of cystic fibrosis or a gene mutation). Thegenetic investigation certainly allows only very limited conclusionsregarding functional disturbances. There are, so far, many hundreddifferent CFTR mutations well-known, which lead to more or less definedclinical images. In the genetic analyses, however, only approx. 15different mutations are examined, which cause only about 80 of all CFillnesses.

7) There was a CF test motivated, in which ATP released by erythrocytesthrough mechanical deformation was to be measured with a luciferaseassay. This is based on the observation of a relationship between CFTRand ATP secretion. (Verloo. P. et al., Pediatric Pulmonology. Suppl.20:72 (2000)).

8) Other methods are based on differences between CF and non-CF inkinetics of some enzymes, e.g. the NADH dehydrogenase of mitochondria(Patent No.: PCT/US8Q/00370, title: Cystic fibrosis detection method),which can be measured e.g. in the lymphocyte homogenate.

9) The enzyme hydrolase of CF patients shows faster inactivatingkinetics than the hydrolase of healthy test subjects (patent No.: U.S.Pat. No. 4,489,788, Title: In vitro diagnosis of cystic fibrosis).

The well-known test procedures are somewhat expensive and have thedisadvantage that they offer little reliability and thus no reliablenewborn screening for mucoviscidosis. Genetic testing is possible withnewborn children, but with heterozygote mutations permits no statementabout the functional extent of the CFTR defect. The sweat test ispracticable starting from the 4th month of life and is connected withsubstantial fluctuations. The measurement of the immunoreactive trypsinand the measurement of the albumin in the meconium are not veryreliable.

By a diagnosis accomplished as early as possible—most effectively withthe newborn child—a purposeful treatment starting from the first lifeday could improve the health situation of the patients, and thus clearlyincrease the life expectancy, and in this case the serious,illness-caused anatomical changes would develop less strongly.

Since the standard techniques do not permit an early diagnosis of cysticfibrosis, CF illnesses can be surely recognized only at a late point intime. In clinical practice, babies in justified cases of suspicion areespecially treated with antibiotics, which causes expense and can leadto health impairments. With the heterozygous CF mutation, it is possiblethat the illness in its varying degrees of severity manifests itselffirst in youth or even later in adulthood excluding successful earlytreatment. Furthermore, there are clinical pictures, for example certainpancreatic illnesses, that are supposed to be associated with mutationsof the CFTR gene.

It is the objective of the invention to provide a suitable method toreliably detect illnesses that are based on defects of the CFTR,especially cystic fibrosis, as early as possible.

This objective is achieved by a diagnosis method in accordance withclaims 1 through 3 that are suitable to identify an illness which isbased on a defect of the CFTR through cell lysis of non-defective cells.According to claim 2 the identification is possible by prevention of theCFTR-dependent volume regulation by, for example, blockade of the ATPrelease, and according to claim 3 through activation (opening) ofCFTR-dependent ion channels of the non-defective cells.

Example of a test procedure in accordance with claim 2 for blood cells:

A non-defective cell is able to control and regulate its cell volume byinterlinking mechanisms during the influx and the efflux of ions andwater under physiological conditions. In non-defective blood cells,after application of a 155 mm potassium chloride medium chloride flowsinto the cell, inter alia through the CFTR. The invention in accordancewith claim 2 is based on the idea to inhibit the regulatory volumedecrease (RVD) by influencing—for example by blockade—the CFTR-dependentATP-transporting channels expressed in the cytomembrane. Inflowingpotassium and chloride ions accumulate in the functional non-defectivecell, which, because of the accompanying water inflow, leads to lysis ofthe blood cells, while a lysis of defective blood cells that lack theinward transport of the ions does not occur. This is especially the casewith CF blood cells, which due to the CFTR defect have no influx ofchloride ions and whose RVD is independent of CFTR. Therefore, nopositive pressure can build up in the cell; the blood cells withdefective ion channels, especially the blood cells of CF patientsundergo no lysis.

In patients suffering from mucoviscidosis with a homozygous ΔF508mutation, the CFTR is not in the cytomembrane. With heterozygousmutations and other mutations than ΔF508, small quantities of functionalCFTR can be in the membrane that transport chloride and are involved involume regulation. The quantity of the functional CFTR molecules in thecytomembrane is inversely proportional to the severity of the illness.The CFTR dependent RVD is inhibited by the blockade of the CFTRdependent ATP transporting channels. The contribution of the CFTRdependent RVD to the entire RVD is proportional to the quantity of thefunctional CFTR molecules within the membrane. The more CFTR in themembrane, the bigger the contribution of the CFTR dependent RVD is tothe entire RVD and the greater is the hemolysis after blockade of theCFTR dependent RVD. Therefore, the degree of the hemolysis afterblockade of the CFTR dependent RVD is a measurement of the functionalCFTR molecules in the cytomembrane. Because the quantity of thefunctional CFTR molecules in the cytomembrane is inversely proportionalto the severity of the illness in mucoviscodosis patients, the degree ofthe hemolysis after blockade of the CFTR dependent RVD is a measurementfor the severity of the illness.

Examinations of the blood of healthy people, mucoviscidosis patients,and blood samples taken postnatal from the umbilical cord of newborns(umbilical cord blood) confirms the easy practicability and thesignificance of the test procedure, in which Gd³⁺ is used for blockadeof the volume regulation. The statistical evaluation of the pastfindings of an investigation with 22 CF patients and 87 healthyvolunteers as well as 59 newborns (using umbilical cord blood) shows anunambiguous increase of the degree of hemolysis after addition of Gd³⁺with the healthy volunteers and with the umbilical cord-blood. With theCF patients, only a slight increase of the degree of hemolysis isrecorded after Gd³⁺ application. Among the 87 healthy volunteers, 75were identified as non-CF, i.e., true negative (86%); and 12 wereidentified as CF, i.e., false positive (14%). With the 22 CF patients,18 were identified as CF, i.e., truly positive (82%), and 4 wereidentified as non-CF, i.e., false negative (18%). 57 of the 59 newbornswere identified as non-CF, i.e. truly negative (97%), and 2 wereidentified as CF, i.e. false positive (3%) (FIG. 1).

Example of a test method according to claim 3 for blood cells:

The objective is accomplished by a diagnosis method according to claim 3that is especially suitable to diagnose CFTR-based illnesses. Thismethod allows the diagnosis of defective blood cells by identifyingnon-defective blood cells by the activation (opening) of certain ionchannels of the non-defective blood cells.

The invention in accordance with claim 3 is based on the idea of evokinga lysis of the non-defective blood cells through activation of certainion channels of the non-defective blood cells, where through anions flowinto the non defective cell, for example: Iodide (I—), bromide (Br—),chloride (Cl—), fluoride (F—), gluconate (dextronic acid-anion),rhodanate (SCN—), and taurine. In defective blood cells these CFTRdependent ion channels don't exist or are not activatable. Therefore,the influx of anions as, for example: iodide, bromide, chloride,fluoride, gluconate, rhodanate and taurine into the defective cell doesnot occur. Then no positive pressure can build up in the cell; the bloodcells with defective CFTR, especially the blood cells of CF Patients arenot subject to lysis.

Since this CFTR dependent ion channel in non-defective blood cellsallows the passage of anions such as for example: iodide, bromide,chloride, fluoride, gluconate and rhodanate as well as taurine, thedifferentiation between defective and non-defective blood cells can alsotake place in 155 mm solutions that contain iodide, bromide, fluoride,gluconate, rhodanate or taurine.

The blood cells with defective ion channels, especially the blood cellsof CF-Patients, are not subject to lysis.

The statistical evaluation of the examination findings among 48 CFpatients and 148 healthy volunteers as well as 82 newborns (umbilicalcord-blood) shows an unambiguous increase of the degree of hemolysisafter addition of sDIDS,(4-amino-4′-isothiocyanato-stilbene-2,2′-disulfonic acid, herein afterreferred to as sDIDS), among the healthy volunteers and with theumbilical cord-blood. With the CF patients, only a slight increase ofthe degree of hemolysis can be recorded after sDIDS application. Amongthe 148 healthy volunteers, 119 were identified as non-CF, i.e. truenegative, 80.4%), and 29 were identified as CF, i.e. false positive,19.6%). Among the 48 CF patients, all 48 were identified as CF, i.e.true positive, 100%). 80 were identified among the 82 newborns asnon-CF, i.e. true negative, 97.5%), and 2 were identified as CF, i.e.false positive, 2,5%), FIG. 2.

The activation of certain CFTR dependent ion channels, which allows thepassage of anions such as, for example: iodide, bromide, chloride,fluoride, gluconate, rhodanate and taurine, is possible with a substancethat originates from DIDS(4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid, sodium salt)having undergone a special treatment. DIDS is dissolved in DMSO (0.1 M)and stored for 4 weeks at 4° C. in the refrigerator. Through thisstorage of DIDS, a hydrolysis-product of DIDS emerges through theresidual water contained within the DMSO. Analysis of this solution withmass spectrometric methods shows that besides DIDS, also4,4′-diamino-stilbene-2,2′-disulfonic acid (DADS) and4-amino-4′-isothiocyanato-stilbene-2,2′-disulfonic acid exist in thesolution (FIG. 3).

Through partial hydrolysis of DIDS, the4-amino-4′-isothiocyanatostilbene-2,2′-disulfonic acid, H₂S and CO₂develops (FIG. 4):4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid, Na⁺-salt(DIDS)+2H₂O=>4-amino-4′-isothiocyanato-stilbene-2,2′-disulfonic acid,Na⁺-salt+H₂S+CO₂.

Storage of 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid(SITS) in hydrous DMSO at 4° C. for four weeks leads to hydrolysisproducts as well, so that also DADS and4-amino-4′-isothiocyanato-stilbene-2,2′-disulfonic acid are to be foundin such a solution as well as SITS. Through partial hydrolysis of SITS,4-amino-4′-isothiocyanato-stilbene-2,2′-disulfonic acid and acetic acidare to be found (FIG. 5):4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid,(SITS)+H₂O=>4-amino-4′-isothiocyanato-stilbene-2,2′-disulfonic acid+acetic acid.

4-amino-4′-isothiocyanato-stilbene-2,2′-disulfonic acid is denominatedsDIDS for the purpose of the present invention.

Also the product of the partial hydrolysis of4,4′-diisothiocyanatodihydrostilbene-2,2′-disulfonic acid, sodium salt(H₂DIDS), behaves like sDIDS. It differs from sDIDS only in a simplerelationship of the carbon atoms between the phenyl rings in contrast toa double bond in sDIDS.4,4′-diisothiocyanato-dihydrostilbene-2,2′-disulfonic acid. Na⁺-salt(H₂DIDS)+2H₂O=>4-amino-4′isothiocyanato-dihydrostilbene-2,2′-disulfonic acid,Na⁺-salt+H₂S+CO₂

The structure-formula is shown in FIG. 7.

It was described in 1987 by Horobin, Payne and Jakobsen (Horobin R W,Payne J N, Jakobsen P. Histochemical implications of the biologicalproperties of SITS and some related compounds. Journal of Microscopy,1987 April; 146(1):87-96), that SITS and DIDS transform in aqueoussolution through hydrolysis of the acetamido group, e.g. anisothiocyanate group, to4-amino-4′-isothiocyanatostilbene-2,2′-disulfonic acid. This occurs atroom temperature within a week. After three weeks, the DADS has formedthrough hydrolysis of the remaining isothiocyanate group. With storageof DIDS or SITS in DMSO over several days at room temperature, polymersare formed through reaction of the isothiocyanate groups with aminogroups (FIG. 6). In the literature, the polymer formation of the DIDS isdescribed (Schultz B D, Singh A K, Devor D C. BRIDGES R J. Pharmacologyof CFTR chloride channel activity. Physiol Rev. 1999 January; 79 (1Suppl): S109-44s. Review).

During the experimental development of the test, it is shown that theobserved reaction of the red blood cells can not be explained by themechanisms known in the literature. The blood cell reactions are onlyexplainable by the existence of a channel proteins, which is not yetknown in literature. By means of patch-clamp technology, the reversibleactivatibility of a conductivity (channel protein) through sDIDS couldbe observed at a human respiratory tract-epithelium-cell-line (Calu-3)in the whole-cell-configuration (FIG. 8).

In the whole-cell-configuration of the patch-clamp technology, thetransmembrane current of a cell is measured with a pre-determinedpotential. The endocellular potential was maintained for 0.5 seconds ineach case at values between −90 to +10 mV and increased in 10 mVintervals (8.d). The currents arising in a cell with this voltage rangeare presented in 8 a-8 c. In 8 a, the base current of an untreated cellis shown (control). Through treatment with sDIDS, the conductivity ofthe cytomembrane of this cell increases together with the current (8 b).After sDIDS was removed from the experiment (post control), theconductivity of the cytomembrane of this cell decreases, together withthe current (8 c). In 8 e, the current is plotted against the appliedpotential, so that the slope of the curve is a measurement of theconductivity.

The novel channel protein enables ions such as, for example: iodide,bromide, chloride, fluoride, gluconate, rhodanate and taurine to pass.Moreover, this channel protein is dependent on CFTR, since in bloodcells of CF patients with sDIDS no conductivity of ions such as, forexample: is iodide, bromide, chloride, fluoride, gluconate, rhodanateand taurine, can be activated.

Here this conductivity is denominated as “SDIDS activatable anionchannel”, abbreviated SDAC, and hereinafter named SDAC.

The past appraisal of blood of healthy persons, mucoviscidosis patientsand postnatal umbilical cord blood samples of newborns (umbilicalcord-blood) confirm the easy practicability and the good explanatorypower of the test method according to the invention.

The method according to the invention is preferably practiced with bloodcells. The use of reticulocytes and young erythrocytes is especiallyadvantageous.

Reticulocytes are the progenitor cells of the red blood corpuscles,erythrocytes. They are produced in the bone marrow and are namedreticulocytes from the moment they enter the blood circulation. Theypossess cell organelles and membrane transport systems. Afterapproximately three days, the reticulocytes mature into youngerythroytes by loss of their cell organelles and a part of theirmembrane transport systems. Erythrocytes stay in the blood circulationapproximately 100 days and continually lose, in the course of this time,their membrane bound proteins. Since erythrocytes have no cell nucleus,the new synthesis of proteins is not possible. The portion of thereticulocytes in the whole blood amounts to 0.4 to 2% of the blood cellsin adults and up to 10% in newborns.

The method according to the invention—influencing the volume regulationof red blood cells by blocking transmembrane proteins or initiating ahigh volume increase and lysis of the non-defective blood cells by anactivation of CFTR-dependent ion channels—can be applied for amultiplicity of further examination methods for identifying otherchannel defects as, for example, the Bartter Syndrome. The BartterSyndrome is a hereditary, kidney damaging illness, that can lead toparalysis, overhydration and general weakening of the body.

Compared to standard methods the method according to the invention hasthe advantage that it is fast and easy applicable with high samplethroughput and thus is cost efficient. The sample material, in the formof small quantities of patient blood, is easily available and can beobtained and processed with the common facilities and instruments of ahospital laboratory. With postnatal umbilical blood, newborn screeningcan take place without invasive action.

Preferred embodiment according to method 1:

In accordance with the invention, first blood cells are extracted fromwhole blood by centrifugation. The blood cells are washed once inisotonic salt solution and are afterwards resuspended in a solutionconsisting of 155 mM KCl, 10 mM HEPES-Puffer(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), (pH 7.4) and 100mM GdCl₃. This solution is incubated for 60 min under agitation at 37°C. and afterwards the released hemoglobin in the solution is determined.The reticulocytes and the new red blood corpuscles from the blood ofhealthy volunteers burst and release their hemoglobin; they undergohemolysis under the specified conditions. The hemolysis is determined byvisual examination and is quantified by absorption measurement with awave length of 546 nm. In addition, a reticulocyte count can also becarried out. Performing the method by each individual practicing doctor,who possibly doesn't have the equipment of a hospital laboratory,thereby becomes practicable.

Through the described use of the Gd³⁺ as inhibitor of the ATP release,an affordable and easy to handle chemical is provided. The test candeliver a reliable result within an hour. The working time and materialexpenditure are especially low in this case.

By choosing the conditions of the preferred embodiment of the methodaccording to the invention, chloride ions flow into the blood cell.Healthy blood cells react thereon with an autocrine signal-cascade, thatis started by the CFTR dependent ATP release and leads to the deliveryof osmolytes. Influx and Efflux are at equilibrium. This CFTR dependentATP release can be inhibited through the addition of the chemicalgadolinium chloride (GdCl₃), which results in an accumulation ofchloride within the cells. Alternatively, also other inhibitors of thisATP release, for example lanthanum ions (La³⁺) in the form of lanthanumchloride (LaCl₃) and other inhibitors of stretch activatible cationchannels, stretch activatable cation channel, SAC, allow the start ofthis ATP release. The water influx associated with the ion influx leadsto bursting of the healthy blood cells. The hemolysis can be detectedphotometrically.

Preferred embodiment according to method 3:

The method according to method 2 is carried out analogously to theexample according to method 1, only the blood cells are resuspended in asolution consisting of 155 mM KCl, 10 mM HEPES-Puffer (pH 7.4) and 100μM sDIDS. This solution is incubated for 60 min under agitation at 37°C. and afterwards the released hemoglobin in the solution is determined.

Through the selected conditions of the preferred embodiment of themethod according to the invention, chloride ions flow into non-defectiveblood cells via the anion channel activated by sDIDS into the bloodcell. That leads to the accumulation of chloride ions in the blood celland the water influx associated ion influx leads the good blood cell toburst. The hemolysis can be detected photometrically. This procedure mayalso be carried out with 10 mM HEPES-buffered solution (pH 7.4), thatcontains 155 mM iodide, bromide, fluoride, gluconate, or rhodanate asanions.

The application of the CF tests is also possible in whole blood.Therewith the test method achieves greater application possibilities,since it can be carried out even more simply and quickly.

A particular advantage of the described tests lies in the possibility toprovide for the first time, to distinguish a healthy CFTR proteinfunctionally from a mutated, sick CFTR protein, as well as to reliablydetect heterozygous CFTR mutations, that lead to physiological changes.In comparison to genetic analysis, the method according to the inventionis a functional test, more economical and allows due to its fastpracticability high test throughputs, which is particularly importantfor screening.

In the context of newborn screenings through the method of theinvention, the consequential damage and thus the consequential expensesof cystic fibrosis can be limited and a meaningful risk assessment canbe carried out for the concerned patient. By the earliest possiblediagnosis, pathological changes of the lung and the digestive tract canbe avoided or at least influenced in its development, since in the firstyear of life severe complications due to intestinal disturbancesfrequently have a lethal outcome. In advanced ages, death as a result oflung failure, including heart failure, through the overstrain of lungcirculation increases. In addition, timely commenced therapy and theavoidance of the administration of inappropriate antibiotics allow aclear improvement of the quality of life and consequently an increase ofthe life expectancy of CF Patients.

Shown in the figures:

FIG. 1: Statistical evaluation of the experiments according to claim 2.

FIG. 2: Statistical evaluation of the experiments according to claim 3.

FIG. 3: Mass spectrum of the improperly stored solution of DIDS in DMSO(0.1 M).

FIG. 4: Partial hydrolysis of DIDS

FIG. 5: Partial hydrolysis of SITS

FIG. 6: Polymerization of DIDS and 4-amino-4′isothiocyanato-stilbene-2,2′-disulfonic acid

FIG. 7: 4-amino4′-isothiocyanato-dihydrostilbene-2,2′ disulfonic acid,disodium salt,

FIG. 8: current and voltage ratio of Calu-3 cells, measured with thepatch-clamp-technique in the whole-cell configuration.

1. A method for the diagnosis of CFTR-defects in patients comprising thefollowing steps: obtaining cells from the patient; destabilising CFTRdependent volume regulation; discriminating between CFTR-defective andCFTR intact cells by determining the volume regulation of the CFRTintact cells.
 2. A method for the diagnosis of CFTR defects according toclaim 1, wherein the step of discriminating between CFTR-defective andCFRT intact cells comprises determining the cell volume of the intactand defective cells.
 3. A method for the diagnosis of CFTR defectsaccording to claim 1, wherein the step of discriminating between CFTRdefective and CFTR intact cells comprises determining cell lysis.
 4. Amethod for the diagnosis of CFTR defects according to claim 1, whereinthe step of destabilising CFTR dependent volume regulation comprisesactivating (opening) or inhibiting a CFTR dependent membrane channel. 5.A method for the diagnosis of CFTR defects according to claim 1, whereinthe cells obtained from the patient are blood cells.
 6. A method for thediagnosis of CFTR defects according to claim 3, wherein thenon-defective cells display hemolysis and the CFTR-defective cellsdisplay non-hemolysis.
 7. A method for the diagnosis of CFTR defectsaccording to to claim 6, wherein an optical procedure is used forquantifying the hemolysis.
 8. A method for the diagnosis of CFTR defectsaccording to claim 6, wherein the cells obtained from the patient areblood cells contained in a smear and wherein the hemolysis is quantifiedby reticulocyte count in the smear.
 9. A method for the diagnosis ofCFTR defects according to claim 4, wherein an inhibitor of ATP-releaseis used for inhibiting the volume regulation.
 10. A method for thediagnosis of CFTR defects according to claim 4, wherein an inhibitor forstretch activatable cation channels (SAC) is used for inhibiting thevolume regulation.
 11. A method for the diagnosis of CFTR defectsaccording to claim 10, wherein the inhibitor is Gd³⁺.
 12. A method forthe diagnosis of CFTR defects according to claim
 4. wherein the CFTRdependent membrane channel is activated by means for activating(opening) the CFTR-dependent ion channel SDAC.
 13. A method for thediagnosis of CFTR defects according to claim 12, wherein a stilbenederivative is the activating agent.
 14. A method for the diagnosis ofCFTR defects according to claim 13, wherein4-amino-4′-isothiocyanato-stilbene-2,2′-disulfonic acid or derivativesis the activating agent.
 15. Use of a stilbene derivative for themanufacture of a means for identifying illnesses based on CFTR defects.16. Use of a stilbene derivative or any other activating agent foractivating the CFTR-dependent iori channel SDAC for the manufacture of ameans for diagnosing illnesses based on CFTR defects in a newbornscreening.
 17. A method for the diagnosis of CFTR defects according toclaim
 1. additionally including the step of determining the degree ofseverity of the cystic fibrosis on the basis of quantitativecomparisons.
 18. A method for the diagnosis of CFTR defects according toclaim 2, wherein the cells obtained from the patient are blood cells.19. A method for the diagnosis of CFTR defects according to claim 3,wherein the cells obtained from the patient are blood cells.
 20. Amethod for the diagnosis of CFTR defects according to claim 4, whereinthe cells obtained from the patient are blood cells.